U.S. patent application number 12/285020 was filed with the patent office on 2009-08-13 for droplet-based cell culture and cell assays using digital microfluidics.
Invention is credited to Irena Barbulovic-Nad, Aaron R. Wheeler.
Application Number | 20090203063 12/285020 |
Document ID | / |
Family ID | 40939206 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090203063 |
Kind Code |
A1 |
Wheeler; Aaron R. ; et
al. |
August 13, 2009 |
Droplet-based cell culture and cell assays using digital
microfluidics
Abstract
We introduce a new method for implementing cell-based assays and
long-term cell culture. The method is based on digital
microfluidics (DMF) which is used to actuate nanoliter droplets of
reagents and cells on a planar array of electrodes. DMF method is
sutable for assaying and culturing both cells in suspension and
cells grown on surface (adherent cells). This method is
advantageous for cell culture and assays due to the automated
manipulation of multiple reagents in addition to reduced reagent
use and analysis time. No adverse effects of actuation by DMF were
observed in assays for cell viability, proliferation, and
biochemistry. These results suggest that DMF has great potential as
a simple yet versatile analytical tool for implementing cell-based
assays and cell culture on the microscale.
Inventors: |
Wheeler; Aaron R.; (Toronto,
CA) ; Barbulovic-Nad; Irena; (Toronto, CA) |
Correspondence
Address: |
Ralph A. Dowell of DOWELL & DOWELL P.C.
2111 Eisenhower Ave, Suite 406
Alexandria
VA
22314
US
|
Family ID: |
40939206 |
Appl. No.: |
12/285020 |
Filed: |
September 26, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61064002 |
Feb 11, 2008 |
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Current U.S.
Class: |
435/29 ;
435/288.7; 435/303.1; 435/395 |
Current CPC
Class: |
B01L 2300/0819 20130101;
B01L 2200/0605 20130101; B01F 13/0071 20130101; B01F 13/0076
20130101; C12M 41/00 20130101; B01L 2300/0867 20130101; B01L
2400/0688 20130101; C12M 25/01 20130101; B01L 2300/089 20130101;
C12M 25/08 20130101; B01L 2400/0427 20130101; C12M 23/16 20130101;
C12M 33/00 20130101; C12Q 1/02 20130101; B01L 3/502792 20130101;
G01N 33/54386 20130101; B01L 2200/027 20130101 |
Class at
Publication: |
435/29 ; 435/395;
435/288.7; 435/303.1 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; C12N 5/02 20060101 C12N005/02; C12M 1/34 20060101
C12M001/34; C12M 1/00 20060101 C12M001/00 |
Claims
1. A digital microfluidic based method of performing any one or
both of cell assays and cell cultures, comprising the steps of: a)
providing a digital microfluidic device having an array of
actuating electrodes formed on a substrate surface, a coating
having a working surface coating the substrate surface and array of
actuating electrodes, an actuating electrode controller for
exciting or de-exciting said actuating electrodes for translating
liquid droplets over said working surface; b) dispensing one or
more first droplets containing a suspension of at least one kind of
cells onto one or more first positions on a working surface of the
digital microfluidic device above the array of actuating electrodes
and substrate surface, and dispensing one or more second droplets
containing any one of at least one chemical reagent, at least one
biochemical reagent, at least one biological reagent, and any
combination thereof onto one or more second positions on the
working surface; c) translating each of the one or more first and
second droplets to a corresponding third position on the working
surface such that they substantially mix to form one or more
secondary droplets; d) incubating the one or more secondary
droplets; and e) analyzing the one or more secondary droplets to
identify products produced by incubation of the one or more
secondary droplets.
2. The method according to claim 1 wherein some or all of said one
or more secondary droplets is split into at least two smaller
secondary droplets either after step c) or after step d), and
wherein step e) is conducted on the at least two smaller secondary
droplets.
3. The method according to claim 1 wherein any one or combination
of said one or more first and second droplets is split into at
least two smaller droplets during any one of steps b) and c) of the
method.
4. The method according to claim 1 wherein said one or more first
droplets contain a single cell.
5. The method according to claim 1 wherein step e) of analyzing
said one or more secondary droplets is performed by detecting
signals emitted from the secondary droplets using a device capable
of detecting a signal from the secondary droplets selected from the
group consisting of optical sensors, optical detectors comprising a
light source and a photodetector, optical detectors that measure
any one or combination of absorbance, fluorescence,
epifluorescence, and chemiluminescence, UV light detectors,
radiometric detectors, any one of scanning, imaging, and confocal
microscopy detectors, CCD cameras, microplate readers.
6. The method according to claim 1 wherein in the step e) said one
or more secondary droplets are translated to one or more selected
positions on said working surface for analysis or the said one or
more secondary droplets are removed from the working surface and
analyzed externally.
7. The method according to claim 1 wherein prior to step b)
including modifying one or more pre-selected positions on said
working surface to produce one or more cell culture sites wherein
said one or more pre-selected positions are located such that each
cell culture site is accessible to droplets being translated by
said electrode array, and wherein adherent cells adhere to said
plurality of cell culture sites.
8. The method according to claim 7 wherein in step c) said one or
more first droplets optionally mixed with one or more second
droplets contain adherent cells and are translated to said one or
more cell culture sites and incubated there to allow cell
attachment thereto, after which said one or more second droplets
are translated onto said one or more cell culture sites such that
they substantially mix with droplets with submersed adhered
cells.
9. The method according to claim 7 wherein in step c) said one or
more first droplets optionally mixed with one or more second
droplets contain adherent cells and are translated onto said one or
more cell culture sites and incubated there to allow cell
attachment thereto, after which said one or more second droplets
are translated onto said one or more cell culture sites such that
the droplet on the cell culture site is replaced by the second
droplet such that the cells attached to the cell culture site are
submersed in said second droplet.
10. The method according to claim 7 wherein said one or more
pre-selected positions are modified by depositing a bio-substrate
thereon.
11. The method according to claim 10 wherein said bio-substrate is
deposited using any one of microprinting and microstamping.
12. The method according to claim 10 wherein said bio-substrate is
produced from cell specific constituents.
13. The method according to claim 12 wherein said cell specific
constituents are extracellular matrix proteins.
14. The method according to claim 13 wherein said extracellular
matrix proteins include any one of fibronectin, laminin, collagen,
elastin and any combination thereof.
15. The method according to claim 12 wherein said cell specific
constituents are synthetic molecules comprised of one of
poly-L-lysine, poly-D-lysine and any combination thereof.
16. The method according to claim 7 wherein said one or more
pre-selected positions on said working surface include any one of a
hydrophobic layer and a dielectric layer, and wherein said cell
culture sites are produced using any one or combination of plasma
treatment, hydrophobic layer etching, dielectric layer etching,
electrode etching and stamping.
17. The method according to claim 1 wherein said at least one
second droplet contains said biological agent only, said biological
agent being a cell culture medium, and wherein said incubated
secondary droplet includes cells cultured in suspension or adherent
cells, and wherein when said cells are adherent cells, including
modifying one or more pre-selected positions on said working
surface to produce one or more cell culture sites wherein said one
or more pre-selected positions are located such that each cell
culture site is accessible to droplets being translated by said
electrode array.
18. The method according to claim 17 including periodically
dispensing additional droplets containing the cell culture medium
and translating the additional droplets to said corresponding third
positions to mix with, or displace and replace, said one or more
secondary droplets.
19. The method according to claim 1 wherein said at least one
second droplet contains each one of or any combination of said
chemical agent and said biochemical agent only, said chemical agent
and biochemical agent being cell assay reagents.
20. The method according to claim 1 wherein said at least one
second droplet contains a combination of said biological agent and
each one of, or any combination of, said chemical agent and said
biochemical agent only, said chemical agent and biochemical agent
being cell assay reagents.
21. The method of performing a cell assay as claimed in claim 1
wherein step b) is performed by dispensing said one or more first
and second droplets from sources external to said digital
microfluidic device.
22. The method of performing a cell assay as claimed in claim 21
wherein the external source is selected from the group consisting
of pipettes, robotic dispensers, microprinters and microstamps.
23. The method of performing a cell assay as claimed in claim 1
wherein step b) is performed by dispensing said first and second
droplets from sources integrated as part of said digital
microfluidic device, said sources being in flow communication with
said working surface.
24. The method of performing a cell assay as claimed in claim 23
wherein said sources integrated as part of said digital
microfluidic device are liquid reservoirs.
25. The method of performing a cell assay as claimed in claim 24
wherein said liquid reservoirs are formed on said working surface
above some of said actuating electrodes which are modified to act
as said liquid reservoirs.
26. The method of performing a cell assay as claimed in claim 1
wherein step b) is performed by dispensing said one or more first
and second droplets from sources integrated as part of a cartridge
assembled with a said digital microfluidic device, said sources
being in flow communication with said working surface.
27. The method according to claim 1 wherein said suspension of
cells is a combination of cells, a suspension medium, and a
non-ionic surfactant.
28. The method according to claim 27 wherein said suspension medium
is selected to facilitate cell-containing droplet actuation by
preventing non-specific adsorption of cells and proteins to device
surfaces.
29. The method according to claim 27 wherein the suspension of
cells is a combination of cells and a suspension medium selected
from the group consisting of block copolymers formed from
poly(propylene oxide) and poly(ethylene oxide), pluronic F68,
pluronic F127, hydrophilic polymers, sodium bicarbonate, phosphate
buffered saline (PBS), HEPES, and other biological buffers, and
cell culture medium selected from the group consisting of balanced
salt solutions, nutrient mixtures, basal media, complex media,
serum free media, insect cell media, virus production media, serum,
fetal bovine serum, serum replacements, antibiotics, antimycotics,
and any combination thereof, and any combination thereof.
30. The method according to claim 27 wherein the suspension of
cells is a combination of cells, phosphate buffered saline, and
pluronic F68.
31. The method according to claim 1 wherein said chemical,
biochemical and biological reagents are selected from the group
consisting of chemicals, biochemicals, drugs, drug lead compounds,
toxins, surfactants, transfection reagents, supplements,
anti-clumping agents, streptavidin, biotin, antibody production
enhancers, antibodies, antibody ligands, nucleic acids, nucleic
acid binding molecules, enzymes, proteins, viruses, cell process
agonists or antagonists, labeling agents, fluorescent dyes,
fluorogenic dyes, viability dyes, calcein AM, quantum dots, nano
particles, Tween 20, and ethidium homodimer-1, block copolymers
formed from poly(propylene oxide) and poly(ethylene oxide),
pluronic F68, pluronic F127, hydrophilic polymers, sodium
bicarbonate, phosphate buffered saline (PBS), HEPES, and other
biological buffers, and cell culture medium selected from the group
consisting of balanced salt solutions, nutrient mixtures, basal
media, complex media, serum free media, insect cell media, virus
production media, serum, fetal bovine serum, serum replacements,
antibiotics, antimycotics, and any combination thereof, and any
combination thereof.
32. The method according to claim 1 wherein the cells in the
suspension of cells include primary/isolated or
transformed/cultured cells selected from the group consisting of
prokaryotic and eukaryotic cells, animal cells (blood cells, human
leukemia cells, lymphocytes, beta cells, oocytes, eggs, primary
cells, primary bone marrow cells, stem cells, neuronal cells,
endothelial cells, epithelial cells, fibroblasts), insect cells,
plant cells, bacterial cells, archebacterial cells.
33. The method according to claim 1 wherein the cells in said
suspension of cells have a density less than about 1.times.10.sup.8
cells/mL.
34. The method according to claim 1 wherein said working surface
includes one or more hydrophilic areas and including translating a
primary droplet over said one or more hydrophilic areas, said
primary droplet having a base area larger than said one or more
hydrophilic areas whereby a first smaller droplet is removed from
said primary droplet and remains behind on said one or more
hydrophilic areas, and wherein said primary droplet is any one of
said one or more first, second and secondary droplets.
35. The method according to claim 34 including translating
additional one or more primary droplets over said one or more
hydrophilic areas already containing a said first smaller droplet,
whereby the first smaller droplet is replaced by a second smaller
droplet from said additional primary droplet.
36. The method according to claim 34 wherein said one or more
hydrophilic areas are cell culture sites.
37. The method according to claim 9 wherein after incubating said
secondary droplet in step d), including translating one or more
droplets containing a washing solution over one or more cell
culture sites to replace an incubation media and wash cells from
the incubation media, then translating one or more droplets
containing a cell dissociation agent to said one or more cell
culture sites, and incubating in order to detach the cells adhered
to said one or more cell culture sites, then mixing one or more
droplets containing the dissociation agent and said detached cells
with one or more droplets containing any one of at least one
dissociation-blocking agent, a biological reagent, a chemical
reagent, a biochemical reagent, and any combination thereof,
wherein a biological reagent can be cell culture media, whereby
forming a secondary droplet with re-suspended cells, and wherein
said re-suspended cells can be optionally further assayed.
38. The method according to claim 37 including mixing said
secondary droplet with re-suspended cells or any droplet split
therefrom with one or more droplets containing cell culture media
and translating at least some cells mixed with the cell culture
media to at least one new cell culture site to seed a new
generation of cells.
39. The method according to claim 1 conducted in a substantially
sterile chamber.
40. The method according to claim 39 including controlling and
regulating conditions in the sterile chamber including humidity,
temperature and atmosphere.
41. The method according to claim 1 performed in a multiplexed
mode, wherein i) said step of dispensing one or more first droplets
includes dispensing a plurality of first droplets each containing a
suspension of at least one kind of cells onto a plurality first
positions on the working surface, ii) and wherein said step of
dispensing one or more second droplet includes dispensing a
plurality of second droplets onto a plurality of second positions
on the working surface, iii) and wherein step c) includes
translating the plurality of first droplets to a corresponding
plurality of third positions on said working surface, and
translating the plurality of second droplets to said corresponding
plurality of third positions in such a way that at least one second
droplet is translated to each of said third positions such that
they substantially mix with the first droplets at each third
position to form a corresponding plurality of secondary
droplets.
42. The method according to claim 41 wherein said plurality of
second droplets is equal to said plurality of first droplets so
each secondary droplet includes a first and second droplet mixed
together.
43. The method according to claim 41 wherein said plurality of
second droplets is a multiple of N times said plurality of first
droplets so each secondary droplet includes N second droplets mixed
with a first droplet.
44. The method according to claim 41 wherein each of said plurality
of second droplets includes a plurality of reagents.
45. The method according to claim 41 wherein step b) is performed
simultaneously for each of said plurality of first and second
droplets.
46. The method according to claim 41 wherein step b) is performed
sequentially for each of said plurality of first and second
droplets in a certain order defined by a cell assay protocol.
47. The method according to claim 41 wherein step c) is performed
simultaneously for each of said plurality of first and second
droplets.
48. The method according to claim 41 wherein step c) is performed
sequentially for each of said plurality of first and second
droplets in a certain order defined by a cell assay protocol.
49. The method according to claim 41 wherein step d) is performed
simultaneously for each of said secondary droplets.
50. The method according to claim 41 wherein step d) is performed
sequentially for each of said secondary droplets in a certain order
defined by a cell assay protocol.
51. The method according to claim 41 wherein step e) is performed
simultaneously for each of said secondary droplets.
52. The method according to claim 41 wherein step e) is performed
sequentially for each of said secondary droplets in a certain order
defined by a cell assay protocol.
53. The method according to claim 41 wherein each of said plurality
of first droplets include cell suspensions identical to the cell
suspensions in the rest of said first droplets, and wherein step i)
is performed by dispensing the plurality of first droplets from one
source.
54. The method according to claim 41 wherein each of said plurality
of first droplets include cell suspensions different to the cell
suspensions in the at least one of said first droplets, and wherein
step i) is performed by dispensing the plurality of first droplets
from a corresponding plurality of sources, each of said plurality
of sources having a cell suspension different to the rest of the
cell suspensions.
55. The method according to claim 41 wherein each of said plurality
of second droplets include reagents identical to the reagents in
the rest of said second droplets, and wherein step ii) is performed
by dispensing the plurality of second droplets from one source.
56. The method according to claim 41 wherein each of said plurality
of second droplets include reagents different to the reagents in
the at least one of said second droplets, and wherein step ii) is
performed by dispensing the plurality of second droplets from a
corresponding plurality of sources, each of said plurality of
sources having a reagent different to the rest of the reagents in
the other sources.
57. The method according to claim 41 wherein prior to step b)
including modifying a plurality of pre-selected positions on said
working surface to produce a plurality of cell culture sites
wherein said plurality of pre-selected positions are located such
that each cell culture site is accessible to droplets being
translated by said electrode array, and wherein adherent cells
adhere to said plurality of cell culture sites.
58. The method according to claim 57 wherein in step c) said
plurality of first droplets optionally mixed with plurality of
second droplets contain adherent cells and are translated to said
plurality of cell culture sites and incubated there to allow cell
attachment thereto, after which said plurality of second droplets
are translated onto said plurality of cell culture sites such that
they substantially mix with droplets with submersed adhered
cells.
59. The method according to claim 57 wherein in step c) said aid
plurality of first droplets optionally mixed with said plurality of
second droplets contain adherent cells and are translated onto said
aid plurality of cell culture sites and incubated there to allow
cell attachment thereto, after which said aid plurality of second
droplets are translated onto said aid plurality of cell culture
sites such that the droplet on the cell culture site is replaced by
the second droplet such that the cells attached to the cell culture
site are submersed in said second droplet.
60. The method according to claim 1 wherein steps b), c), d) and e)
are conducted according to a selected cell assay or cell culture
protocol under control of a computer controller interfaced to said
digital microfluidic device.
61. A digital microfluidic device for conducting one or both of
cell assays and cell culture, comprising: a first substrate having
a first substrate surface; an array of actuating electrodes formed
on the first substrate surface; at least one dielectric layer
formed on the first substrate surface covering each actuating
electrode such that the actuating electrodes are electrically
insulated from one another; and at least one reference electrode,
wherein each actuating electrode is proximal to at least one of the
reference electrodes; an electrode controller capable of
selectively exciting or de-exciting actuating electrodes for
translating liquid droplets across a surface of the dielectric
layer; one or more first reservoirs in flow communication with the
surface of said dielectric layer for holding at least one
suspension of cells and one or more reagent reservoirs in flow
communication with the surface of said dielectric layer for holding
one or more cell assay reagents, cell culture reagents; and
dispensing means for dispensing droplets of said at least one
suspension of cells and droplets of said at least one cell assay
reagents, cell culture reagents onto said surface of said
dielectric layer; and a computer controller interfaced to said
dispensing means and said electrode controller and being programmed
to dispense droplets of the suspension of cells and droplets of
said one or more cell assay reagents, cell culture reagents onto
said surface of said dielectric layer and translating them over
said array of actuating electrodes for mixing and optionally
splitting said droplets in selected positions on said array of
actuating electrodes to form one or more secondary droplets in a
selected order defined by a selected cell assay protocol or cell
culture protocol for which said computer controller is
programmed.
62. The device according to claim 61 wherein said surface of said
dielectric layer includes one or more pre-selected positions having
cell culture sites located thereon, and wherein said one or more
pre-selected positions are located such that each cell culture site
is accessible to droplets being translated by said electrode array,
and wherein adherent cells adhere to said plurality of cell culture
sites.
63. The device according to claim 61 wherein the surface of the
dielectric layer is hydrophobic.
64. A device according to claim 61 further comprising a first
hydrophobic layer formed on said surface of the dielectric layer,
and wherein all droplets are dispensed onto said hydrophobic layer
for translation.
65. The device according to claim 61 including detection and
analyzing means for detecting and analyzing signals from said one
or more secondary droplets to identify products produced by
incubation of the one or more secondary droplets.
66. The device according to claim 65 wherein said detection and
analyzing means is selected from the group consisting of optical
sensors, optical detectors comprising a light source and a
photodetector, optical detectors that measure absorbance,
fluorescence, epifluorescence, chemiluminescence, UV light
detector, radiometric detector, scanning, imaging, and confocal
microscopy detectors, CCD cameras, and microplate readers.
67. The device according to claim 61 further including a second
substrate and a hydrophobic layer on a surface thereof, wherein the
second substrate is in a spaced relationship to the first plate
thus defining a space between the first and second substrates
capable of containing droplets between the hydrophobic layer of the
second substrate and the dielectric layer on the first
substrate.
68. A device according to claim 67 wherein the second substrate is
substantially transparent.
69. The device according to claim 61 wherein said array of
actuating electrodes is an array of substantially coplanar
actuating electrodes formed on the first substrate surface.
70. The device according to claim 69 wherein said at least one
reference electrode is a grid of generally elongate reference
electrodes formed in-between, and electrically insulated from, the
actuating electrodes in the array of substantially coplanar
actuating electrodes.
71. The device according to claim 61 further including a second
substrate and a hydrophobic layer on a surface thereof, wherein the
second substrate is in a spaced relationship to the first plate
thus defining a space between the first and second substrates
capable of containing droplets between the hydrophobic layer of the
second substrate and the dielectric layer on the first substrate,
and wherein said hydrophobic layer includes one or more
pre-selected positions having cell culture sites located thereon,
and wherein said one or more pre-selected positions are located
such that each cell culture site is accessible to droplets being
translated by said electrode array, and wherein adherent cells
adhere to said plurality of cell culture sites.
72. The method according to claim 41 wherein said plurality of
second droplets contain said biological agent only, said biological
agent being a cell culture medium, and wherein said plurality of
incubated secondary droplets include cells cultured in suspension
or adherent cells, and wherein when said cells are adherent cells
including modifying a plurality of pre-selected positions on said
working surface to produce a plurality of cell culture sites
wherein said plurality of pre-selected positions are located such
that each cell culture site is accessible to droplets being
translated by said electrode array.
73. The method according to claim 72 including periodically
dispensing a plurality of additional droplets containing the cell
culture medium and translating the plurality of additional droplets
to said corresponding plurality of third positions to mix with, or
displace and replace, said plurality of secondary droplets.
Description
CROSS REFERENCE TO RELATED U.S. APPLICATIONS
[0001] This patent application relates to, and claims the priority
benefit from, U.S. Provisional Patent Application Ser. No.
61/064,002 filed on Feb. 11, 2008, in English, entitled
DROPLET-BASED CELL ASSAYS, and which is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to droplet-based cell assays
and/or cell culture using digital microfluidics, and more
particularly, the present invention relates to devices and methods
used with those devices for performing cell assays and/or cell
culture.
BACKGROUND OF THE INVENTION
[0003] The cell is the irreducible element of life and is often
studied as a living model of complex biological systems. Cell-based
assays are conventionally performed in well plates that enable
simultaneous analysis of multiple cell types or stimuli. For such
multiplexed analyses, cells in well plates are often evaluated
using microplate readers, which can be integrated with fluid
handling and other miscellaneous equipment in a robotic analysis
platform. A major drawback of such systems is the expense of the
instrumentation and the experimental consumables (e.g., plates,
pipette tips, reagents, and cells). The latter is a particular
disadvantage for cell-based assays as they are generally more
complex and require larger amounts of reagents than cell-free
assays..sup.1
[0004] Recently, microfluidics has been touted as a solution for
the challenges inherent in conducting multiplexed cell-based
assays..sup.2 The conventional format for microfluidics, which is
characterized by devices containing networks of micron-dimension
channels, allows integration of multiple processes on a single
platform while reducing reagent consumption and analysis time.
There are numerous advantages of using microfluidic based systems
for cell assays, some of which are self-similarity in dimensions of
cells and microchannels (10-100 .mu.m widths and depths), laminar
flow dominance and formation of highly resolved chemical gradients,
subcellular delivery of stimuli, reduced dilution of analytes, and
favorable scaling of electrical and magnetic fields. For the last
ten years, researchers have used microchannels to manipulate and
sort cells, to analyze cell lysates, to assay intact-cell
biochemistry, and to evaluate cell mechanical and electrical
responses. In most of these studies, cells were exposed to one
stimulus or to a limited number of stimuli. There have been just a
few attempts to conduct multiplexed assays as it is difficult to
control many reagents simultaneously in a complex network of
connected channels, even when using microvalve architectures
developed for microfluidic devices..sup.3 Finally, we note that
there have been only a few microfluidic devices integrated to
multiplexed detection instruments such as microplate readers;.sup.4
we believe this will be a necessary step for the technology to
become competitive with robotic screening systems.
[0005] A potential solution to the limitations of the
channel-microfluidic format is the use of "digital" or
droplet-based microfluidics. In digital microfluidics (DMF),
discrete droplets containing reagents are manipulated by
sequentially applying potentials to adjacent electrodes in an
array..sup.5-14 Droplets can be manipulated independently or in
parallel on a reconfigurable path defined by the electrode
actuation sequence, which allows for precise spatial and temporal
control over reagents. As with all microscale techniques,
cross-contamination is a concern for DMF, but this phenomenon can
be avoided by dedicating separate paths for each reagent. DMF has
been used to actuate a wide range of volumes (nL to .mu.L) and,
unlike channel devices, there is no sample wasted in creating small
plugs for analysis. In addition, each droplet is isolated from its
surroundings rather than being embedded in a stream of fluid--a
simple method of forming a microreactor in which there is no
possibility that products will diffuse away. The preservation of
products in a droplet is of great importance in cell assays
targeting molecules secreted from cells into extracellular space.
In addition, droplets provide mostly static fluid conditions
without unwanted shear stress that is inevitable in continuous flow
microfluidics. A further advantage of DMF is its capacity to
generate nanoliter samples by translating droplets through
selective wettability areas on an electrowetting-based
platform..sup.15
[0006] There is currently much enthusiasm for using DMF to
implement multiplexed assays; however, it has only been applied to
a few non-cell assays. To the inventors' knowledge, there are no
reports of the use of DMF to analyze cells. There are a few studies
demonstrating only dispensing and manipulation of droplets
containing cells, cell sorting, and cell concentration on a DMF
platform. WO 2007/120241 A2 entitled "Droplet-Based
Biochemistry".sup.16 discloses dispensing and dividing droplets
containing cells, generating droplets with single cells, detecting
a type of cell, and sorting cells. US20070148763 A1 entitled
"Quantitative cell dispensing apparatus using liquid drop
manipulation".sup.17 describes cell droplet handling, to achieve a
predetermined number of cells. In a journal paper by Fan et
al,.sup.18 dielectrophoresis was used to concentrate neuroblastoma
cells within droplets on a DMF platform.
[0007] It would be very advantageous to provide droplet-based cell
culture and/or assays using digital microfluidics in order to
enable automated cell micro culture and high-throughput screening
ability for cell analysis. DMF would also address some problems
associated with standard culture and assaying in well-plates or in
continuous-flow microfluidic devices.
SUMMARY OF INVENTION
[0008] The present invention provides embodiments of devices and
methods for droplet-based cell culture and assays using digital
microfluidic devices designed to manipulate, operate, and analyze
cell-containing droplets. Cells in a suspension and cell-assay
and/or cell-culture reagents are deposited in the device by either
dispensing them from device reservoirs or dispensing them into the
device using external means (e.g., pipette, robotic dispenser,
etc.). In order to perform an assay with cells in suspension,
cell-containing droplets and reagent-containing droplets are moved
between adjacent electrodes by applying voltages to electrodes.
General assay protocol comprises dispensing and translating
droplets, merging and mixing droplets with cells and reagents at
least once, possible splitting of droplets, incubating cells with
reagents in merged/mixed (and split) droplets at least once, and
detecting signal from cells in merged/mixed (and split) droplets in
the device after final incubation. Using the same DMF techniques,
suspended cells are also long-term cultured and split at regular
time intervals.
[0009] Additionally, DMF devices are designed to culture and assay
adherent cells. After being introduced in a device in suspension,
adherent cells are seeded on cell culture sites (patterned DMF
device surface for cell attachment), where they can be long-term
cultured in droplets, subcultured using standard subculture
protocols, and assayed. Media exchange and regent delivery on cell
culture sites (CSSs) is performed using standard DMF operations:
translating, merging, mixing and splitting droplets. In addition, a
new technique, passive dispensing, is developed for more efficient
delivery of reagents/media from big source droplet translating over
CCSs. By means of DMF and passive dispensing, a first
multigenerational cell culture in a microscale is realized.
[0010] Culture and assay reagents comprise chemical, biochemical
and biological reagents. Droplets contain additives including
pluronics and various hydrophilic polymers to facilitate
cell-containing droplet actuation by preventing non-specific
adsorption of cells and proteins to a device surface.
[0011] In a multiplexed assay, multiple cell-containing droplets
(which may include one kind or multiple kinds of cells) are
manipulated and assayed simultaneously or in a certain sequence
with one or multiple reagents.
[0012] Thus, in an embodiment of the present there is provided a
digital microfluidic based method of performing any one or both of
cell assays and cell cultures, comprising the steps of:
[0013] a) providing a digital microfluidic device having an array
of actuating electrodes formed on a substrate surface, a coating
having a working surface coating the substrate surface and array of
actuating electrodes, an actuating electrode controller for
exciting or de-exciting said actuating electrodes for translating
liquid droplets over said working surface;
[0014] b) dispensing one or more first droplets containing a
suspension of at least one kind of cells onto one or more first
positions on a working surface of the digital microfluidic device
above the array of actuating electrodes and substrate surface, and
dispensing one or more second droplets containing any one of at
least one chemical reagent, at least one biochemical reagent, at
least one biological reagent, and any combination thereof onto one
or more second positions on the working surface;
[0015] c) translating each of the one or more first and second
droplets to a corresponding third position on the working surface
such that they substantially mix to form one or more secondary
droplets;
[0016] d) incubating the one or more secondary droplets; and
[0017] e) analyzing the one or more secondary droplets to identify
products produced by incubation of the one or more secondary
droplets.
[0018] In another aspect of the present invention there is provided
a digital microfluidic device for conducting one or both of cell
assays and cell culture, comprising: [0019] a first substrate
having a first substrate surface; [0020] an array of actuating
electrodes formed on the first substrate surface; [0021] at least
one dielectric layer formed on the first substrate surface covering
each actuating electrode such that the actuating electrodes are
electrically insulated from one another; and [0022] at least one
reference electrode, wherein each actuating electrode is proximal
to at least one of the reference electrodes; [0023] an electrode
controller capable of selectively exciting or de-exciting actuating
electrodes for translating liquid droplets across a surface of the
dielectric layer; [0024] one or more first reservoirs in flow
communication with the surface of said dielectric layer for holding
at least one suspension of cells and one or more reagent reservoirs
in flow communication with the surface of said dielectric layer for
holding one or more cell assay reagents, cell culture reagents; and
[0025] dispensing means for dispensing droplets of said at least
one suspension of cells and droplets of said at least one cell
assay reagents, cell culture reagents onto said surface of said
dielectric layer; and [0026] a computer controller interfaced to
said dispensing means and said electrode controller and being
programmed to dispense droplets of the suspension of cells and
droplets of said one or more cell assay reagents, cell culture
reagents onto said surface of said dielectric layer and translating
them over said array of actuating electrodes for mixing and
optionally splitting said droplets in selected positions on said
array of actuating electrodes to form one or more secondary
droplets in a selected order defined by a selected cell assay
protocol or cell culture protocol for which said computer
controller is programmed.
[0027] A further understanding of the functional and advantageous
aspects of the invention can be realized by reference to the
following detailed description and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Preferred embodiments of the invention will now be
described, by way of example only, with reference to the drawings,
in which:
[0029] FIG. 1 is a top view of a complete digital-microfluidic
device showing three droplet sources: cells, reagent, and dye;
[0030] FIG. 2(a) shows a cross-sectional view of the device of FIG.
1;
[0031] FIG. 2(b) shows a cross sectional view of an alternative
embodiment of the device of FIG. 1 which uses a one-plate
design;
[0032] FIGS. 3(a) to (c) show three frames from a movie wherein a
droplet with cells is dispensed from a reservoir;
[0033] FIG. 4 is a plot of numerically simulated potential drops
across a droplet and a dielectric layer;
[0034] FIG. 5 is a graph of viability and proliferation tests for
cells actuated by digital microfluidics showing no significant
differences between the actuated and non-actuated cells;
[0035] FIGS. 6(a) and (b) are graphs of vitality tests wherein
cells in droplets were actuated, lysed, and analyzed by Matrix
Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS)
showing no major qualitative differences between the (a) actuated
and (b) non-actuated cells;
[0036] FIGS. 7(a) to (f) show sequential images from a movie
depicting a digital microfluidic cell-based assay;
[0037] FIGS. 8(a) and (b) show fluorescent images of droplets with
cells treated with (a) 0% and (b) 0.5% Tween 20 and stained with
viability dyes. (calcein AM and ethidium homodimer-1); in the
droplet (a), almost all cells were live (dead cells in (a) are
marked with small circles), and in the droplet (b), all cells were
dead;
[0038] FIGS. 9(a) and (b) show two dose-response curves for Jurkat
T-cells exposed to Tween 20 (0.002% to 0.5% (v/v)) using (a) a
digital microfluidics assay and (b) a well-plate assay;
[0039] FIG. 10 shows a top view of an embodiment of a DMF device
for multiplexed cell assays which comprises reservoirs for four
different cell suspensions and nine different assay reagents, and a
waste reservoir;
[0040] FIGS. 11(a) to (d) are diagrammatic representations of
seeding adherent cells in a DMF device where (a) shows actively
dispensed droplet of cell suspension translating to a cell culture
site (CCS), (b) shows passively dispensing a droplet of cell
suspension onto the CCS from a source droplet, (c) shows cells in
suspension seeded on the CCS, and (d) shows cell monolayer formed
on the ECM substrate on the CCS;
[0041] FIG. 12 is a diagrammatic representation showing passive
dispensing of a droplet where a source droplet provides a smaller
liquid droplet located on the CCS;
[0042] FIG. 13 shows several examples of the hydrophilic area
positions relative to actuating electrodes and to the source
droplet path;
[0043] FIG. 14 shows a diagrammatic representation showing a
passive washing/exchange process whereby a droplet on a CCS is
replaced by a new droplet;
[0044] FIG. 15 shows a graph of fluorescein fluorescence signal
intensity versus washing cycle to show washing efficiency;
[0045] FIG. 16 shows a digital image of .about.130 mouse fibroblast
cells (NIH-3T3) cultured in a DMF device for 72 h; media was
replenished using passive dispensing/exchange technique every 24 h;
after 72 h cells were stained with calcein AM for viability;
[0046] FIGS. 17(a) to (f) are diagrammatic representations of
subculturing adherent cells in a DMF device in which (a) shows
monolayer of adherent cells cultured on a CCS, (b) washing cells
via passive exchange, (c) delivering a dissociation agent to cells
via passive exchange, (d) detachment of cells after incubation with
a dissociation agent, (e) blocking of a dissociation agent and
resuspending cells via passive exchange, and (f) seeding of cells
resuspended in fresh media on a new CCS;
[0047] FIGS. 18(a) to (d) show diagrammatic representations of
assaying adherent cells in a DMF device where, (a) shows a
monolayer of adherent cells cultured on a CCS in cell culture
media, (b) washing cells and delivering assay reagents to cells via
passive exchange, (c) incubating cells with assay reagents, and (d)
detecting and analyzing cell response to assay stimuli; and
[0048] FIG. 19 shows a DMF device for multiplexed cell assays with
adherent cells using passive dispensing and passive reagent
exchange.
DETAILED DESCRIPTION OF THE INVENTION
[0049] Without limitation, the majority of the systems described
herein are directed to methods and devices for droplet-based cell
assays using digital microfluidics. As required, embodiments of the
present invention are disclosed herein. However, the disclosed
embodiments are merely exemplary, and it should be understood that
the invention may be embodied in many various and alternative
forms.
[0050] The figures are not to scale and some features may be
exaggerated or minimized to show details of particular elements
while related elements may have been eliminated to prevent
obscuring novel aspects. Therefore, specific structural and
functional details disclosed herein are not to be interpreted as
limiting but merely as a basis for the claims and as a
representative basis for teaching one skilled in the art to
variously employ the present invention. For purposes of teaching
and not limitation, the illustrated embodiments are directed to
droplet-based cell assays and culture using digital microfluidics
(DMF).
[0051] As used herein, the term "about" and the symbol ".about.",
when used in conjunction with ranges of dimensions, temperatures or
other physical and/or chemical properties and/or characteristics is
meant to cover slight variations that may exist in the upper and
lower limits of the ranges of dimensions as to not exclude
embodiments whereon average most of the dimensions are satisfied
but where statistically dimensions may exist outside this region.
For example, in embodiments of the present invention dimensions of
a digital microfluidic device are given but it will be understood
that these are not meant to be limiting.
[0052] FIG. 1 shows a top view of a microfluidic device shown
generally at 10 which may be used for droplet-based cell culture
and cell assays using digital digital microfluidics in accordance
with the present invention. Reservoir electrodes 32, 34, and 36
store droplets 42, 44, 46 containing cells, reagent, and dye,
respectively, and are capable of dispensing the liquids onto the
center region 38 of the device. Small volumes of liquids are
dispensed as droplets and translated by applying voltages to
actuating electrodes 14. There is also another reservoir electrode
30 shown in the device in FIG. 1 which may be used as a reservoir
as well.
[0053] FIG. 2(a) is a cross-sectional view of a portion of the
microfluidic device 10 of FIG. 1 showing two adjacent electrodes 14
of the electrode array. Electrodes 14 (10 nm Cr+, 100 nm Au) rest
on a substrate layer 12 and are separated from each other by a
dielectric material 16 (for example 2 .mu.m Parylene-C). The device
can have more than one dielectric layer 16. Located on top of
dielectric material 16 is a hydrophobic layer 18 (for example
Teflon AF, 50 nm). The array of actuating electrodes and exposed
areas of substrate surface are thus covered by a working surface.
Spaced above electrodes 14/dielectric layer 16 is a continuous
reference electrode 22 coated on a substrate layer 24, and a
hydrophobic layer 20 (for example Teflon AF, 50 nm) is coated on
reference electrode 22. Alternatively, another dielectric layer can
be deposited between layers 20, 22. Liquid droplets 42 rest
in-between two hydrophobic layers 18 and 20. Electrodes 14, voltage
source 26, and the continuous reference electrode 22 together form
an electric field, digitally manipulated by controller 28. For
droplet manipulation, reference electrodes 22 are biased to a
potential different from the actuating potential. Commonly used
reference potential is ground.
[0054] In a preferred embodiment of the present invention, the
upper hydrophobic layer 20, reference electrode 22, and substrate
layer 24 are substantially transparent to allow optical analysis of
the assays. Furthermore, layers 20, 22, and 24 are not necessary to
translate droplets.
[0055] While the present invention discusses the two-plate design
of FIG. 2(a), a one-plate design is also possible, as shown in FIG.
2(b). In FIG. 2(b), layers 20, 22, and 24 are removed. Rather than
have a dedicated reference electrode layer 22, the reference
electrode is patterned adjacent to electrodes 14, forming a
continuous grid 52 separated from electrodes 14 by dielectric
material 16. The continuous grid 52 extends in both directions
defining the plane in which electrodes 14 are located.
[0056] Reference electrodes can also be coplanar with the top
surface of the dielectric layer. In a device with multiple
dielectric layers, reference electrodes can be coplanar with the
top surface of any dielectric layer, while being insulated from
actuating electrodes 14. The design of reference electrodes is not
limited to a grid, e.g. they can be in a form of a wire or an array
similarly to electrodes 14.
[0057] FIG. 3 shows three frames from a movie wherein a 150 nL
droplet 42 containing .about.260 cells is dispensed from a
reservoir of a microfluidic device with identical dimensions but
fewer electrodes than the microfluidic device 10 shown in FIG. 1,
wherein cells were labeled with a viability dye, calcein AM, which
fluoresces green.
[0058] FIGS. 7(a) to (f) show sequential images from a movie
depicting a digital microfluidic cell-based assay, wherein a 150 nL
droplet 42 containing .about.525 cells was dispensed (a, 402),
translated (b, 404), and merged (c, 406) with a 150 nL droplet 44
of Tween 20 dispensed (b, 402) from a second reservoir. The merged
droplet was actively mixed (408) on four neighboring electrodes
(d); after 20 min incubation in a humidified environment, the
combined droplet was merged (e, 406) and mixed (e, 408) with a 150
nL droplet 46 containing viability dyes. The final droplet was
incubated (f, 410) for 20 minutes in a humidified environment.
[0059] A sample result of the microfluidic cell-based assay of FIG.
7(f) is shown in FIGS. 8(a) and (b), wherein fluorescent images of
droplets treated with (a) 0% and (b) 0.5% Tween 20. Calcein AM
(green) was used to stain live cells, and ethidium homodimer-1
(red) for dead cells. In the former droplet (a), almost all cells
were live (dead cells in (a) are marked with small circles), and in
the latter (b), all cells were dead.
[0060] While digital microfluidics has been used previously to
manipulate and evaluate a wide range of liquids and reagents, we
report herein the first application of digital microfluidics to
transport, analyze and culture biological cells. Using the
parameters reported in the experimental section (elaborated below),
cell suspensions representing a wide range of concentrations
(including very dense solutions of 1.times.10.sup.8 cells/mL) were
found to be feasible to be actuated by DMF, with no differences
observed in velocity or reliability relative to liquids not
containing cells.
[0061] For example, FIGS. 3(a) to (c) depict a routine operation in
our experiments: dispensing of a 150 nL droplet containing
.about.260 Jurkat T-cells. However, in initial work (with
un-optimized parameters), droplets containing cells were difficult
to manipulate, as cells tended to stick to the surface of the
devices, causing contact line pinning. This problem was overcome by
the use of the non-ionic surfactant, pluronic F68, which when used
as a solution additive, facilitated actuation of suspensions of
cells in all liquids tested (including PBS and complete media
containing 10% fetal bovine serum).
[0062] Pluronics are block copolymers formed from poly(propylene
oxide) (PPO) and poly(ethylene oxide) (PEO), and are commonly used
as surface coatings for preventing non-specific protein adsorption.
In our work, we used pluronics in solution, rather than as a
surface coating; we hypothesize that in this configuration, the
polymer coats cells and proteins in a manner such that their
functionality is retained, but adsorption to hydrophobic surfaces
is minimized. We note that pluronic F68 has been used extensively
in cell-based assays with no evidence for detrimental effects on
cell vitality,.sup.19,20 and it is even used as a constituent in
commercial cell growth media..sup.21 Our experiments support this
trend--Jurkat T-cells incubated in medium containing 0.2% (wt/vol)
F68 for 4 days (humidified incubator, 5% CO2, 37.degree. C.) had
identical growth rates and morphology as cells grown in media
without pluronics. In on-going work, the optimal conditions
(concentration and type of pluronic, etc.) for reducing unwanted
adsorption in DMF are being evaluated; we used F68 for all of the
results reported here.
[0063] A second challenge for using DMF for actuation of cells is
droplet evaporation, which raises the concentration of salts and
other buffer constituents, making the solution hypertonic. In the
work described here, we controlled evaporation by positioning
devices in a humidified atmosphere when not actively manipulating
droplets by DMF. For the duration of the assay experiments (up to a
few hours), such measures prevented significant evaporation, and no
negative effects on cell viability were observed. For culturing
cells, devices were placed in cell culture incubators at 37.degree.
C. and 5% CO.sub.2. The DMF devices may be contained in a sterile,
humidified chamber for the full duration of the assay or cell
culture process (including actuation, incubation, and analysis) or
culture which facilitates long-term cell culture and
examination.
Effects of DMF Manipulation on Cell Vitality.
[0064] Digital microfluidic devices use electrical fields to
actuate droplets, which led us to investigate the effects of
droplet actuation on cell vitality. As described above, droplets
are translated by an energized actuating electrode 14 on a bottom
plate and a reference electrode 22 on a top plate (FIG. 2(a)). It
should be noted that the reference electrode may also be placed on
the bottom plate, as in reference electrode 52 (FIG. 2(b)). Because
of the high conductivity of a droplet 42 of phosphate buffered
saline (PBS) relative to the insulating dielectric layer 16 formed
from Parylene-C, the inventors believe that cells would experience
negligible electrical field upon application of driving potentials.
This hypothesis was supported by a numerical simulation using the
COMSOL Multiphysics 3.3a analysis package. In a simulation, shown
in FIG. 4, in which 100 V was applied between top and bottom
electrodes, the potential drop in the droplet was found to be only
3.73.times.10.sup.-8 V, or 0.00000004% of the applied potential.
Thus, it is contemplated that one would expect to observe modest
effects (if any) on the vitality of suspensions of cells, upon
application of electrical field. These effects were evaluated by
three tests, measuring cell viability, proliferation, and
biochemistry.
[0065] As shown in FIG. 5, the viability of actuated and
non-actuated cells was compared immediately after actuation, and
proliferation was measured after 48-h incubation in a humidified
incubator. There was no significant difference between actuated and
non-actuated cells (P=0.11 for the viability assay, P=0.43 for the
proliferation assay).
[0066] Cell biochemistry was evaluated qualitatively by analyzing
lysates with MALDI mass spectrometry. FIGS. 6(a) and (b) show
spectra of lysates of actuated cells and non-actuated cells,
respectively. From previous studies of protein content in Jurkat
T-cells,.sup.22 we tentatively assigned several peaks, including
heat shock protein (HSP10) 302, macrophage migration inhibitory
factor 304, epidermal fatty-acid binding protein (E-FABP) 306, and
peptidyl-prolyl cis-trans isomerase A 308. As shown, there are no
major qualitative differences between the two spectra, which
suggests that actuation by DMF does not cause catastrophic effects
on cell biochemistry. We note that MALDI-MS is not a quantitative
analysis technique (i.e., peak heights can vary considerably within
multiple spectra of a single sample) The gene expression of T-cells
and other cell types using quantitative PCR or gene microarray
would be more appropriate quantitative techniques.
Cell Phenotype Assays by DMF.
[0067] To illustrate that DMF is compatible with phenotypic assays,
a dose-response toxicology screen was performed using Jurkat
T-cells, shown in FIGS. 7 and 8. Cells were exposed to varying
concentrations of the surfactant, Tween 20 (0.002% to 0.5% (v/v))
(FIG. 7) and then stained with viability dyes (FIG. 8). The
complete assay, from droplet dispensing to the final incubation
with dyes was performed on-chip. 150 nL droplets (.about.1 mm in
diameter) were dispensed via DMF, and after merging and incubation,
resulted in a final .about.450 nL droplet (.about.1.8 mm diameter,
150 .mu.m height). An equivalent assay was implemented in a
384-well plate with the same number of cells (.about.525 cells/well
or droplet) but different sample volume. In the well-plate assays,
5 .mu.L aliquots of each reagent were pipetted into conical wells
(3.3 mm top-, 2 mm bottom diameter) resulting in a final volume of
15 .mu.L (.about.5 mm height) which is in the recommended range for
384-well plates. Hence, well plates required .about.30-fold greater
reagent use than DMF, leading to a much lower cell concentration in
the wells. As described below, this had significant effects on
assay sensitivity.
[0068] A fluorescence microplate reader was used to generate dose
response curves for DMF and well plate assays using identical
settings (FIG. 9, error bars are 1 standard deviation). As shown in
FIG. 9, the DMF assays (a) had much lower background signals than
the well-plate assay (b), resulting in a much larger
signal-to-noise ratio than the well-based assays (b). As a
consequence, the lowest detectable number of live cells in droplets
was .about.10 (a), compared to .about.200 cells in wells (b). The
latter value matches the general limits of detection listed by the
manufacturer for such assays. One consequence of this difference
was the determination of different 100%--lethal concentrations of
Tween 20: .about.0.5% (v/v) from the DMF assay and .about.0.03%
(v/v) from the well plate assay. The true 100%-lethal concentration
was determined empirically by staining cells exposed to varying
concentrations of Tween-20 and counting them using a hemacytometer.
At the concentrations evaluated here, the fluorescence microplate
reader results generated by the digital microfluidic method (a)
were found to be a much better approximation of the empirical value
than the conventional method (b). Thus, in this assay, the
conventional method over-estimates the toxicity of Tween 20 by more
than 15-fold; this is important, as cytotoxicity is widely used by
regulatory agencies in initial screens for determining acceptable
exposure limits, and by the pharmaceutical industry in early drug
discovery.
[0069] Another cause of the improved sensitivity in droplet-based
assays is the high cell concentration in .about.nL droplets. The
same number of cells in .mu.L aliquots results in a much lower
concentration and therefore, lower signal-to-noise ratio. In this
experiment, 525 cells yielded 1.2.times.10.sup.6 cells/mL in
droplets, but only 3.5.times.10.sup.4 cells/mL in wells. In
addition, the cross-sectional density of cells in droplets was
higher because of the slightly smaller droplet diameter (.about.1.8
mm) relative to that of the conical wells (2 mm bottom, 3.3 mm
top). If it is assumed that all cells settled to the bottom of each
well or droplet, then the same number of cells was distributed over
an area that was .about.20% smaller in droplets relative to wells,
resulting in a higher signal. It is possible that all cells
sedimented in droplets (150 .mu.m height), while not all cells
sedimented in wells (.about.5 mm height). If this were the case, it
would obviously contribute to the observed differences in detection
limits.
[0070] It should be noted that while the assay described above
involved dispensing, translating, merging and mixing of droplets,
other embodiments of cell assays and cell culture in DMF devices
can include droplet splitting. Droplet splitting is implemented to
reduce a droplet size, number of cells in a droplet, etc.
[0071] Some cell assays target molecules that cells secrete into
their microenvironment, such as growth factors, signaling
molecules, and metabolic products. Since DMF droplets of cell
suspension are precise, confined volumes where all cell products
are preserved, they are ideal microenvironment for extracellular
biochemistry assays. In these assays, signal is detected from a
suspension medium rather than cells. Suspension medium can be
analyzed by immunoassays or other means. Droplets of cell
suspension can alternatively be removed from a DMF device and
analyzed externally.
[0072] The results presented above demonstrate assaying population
of cells of one kind; nevertheless, it is also possible to assay
droplets containing multiple kinds of cells (e.g., different cell
types, or different phenotypes of the same cell type). Droplets
with multiple kinds of cells can be generated by either dispensing
them from reservoirs containing the same mixed population of cells,
or by combining droplets containing one or several kinds of cells.
Combining droplets, merging and mixing, results in larger droplets
which can be split in droplets of desired size.
[0073] Concentration of cells in a droplet can be controlled by the
concentration of cells in a source (a device reservoir or an
external reservoir) or by combining droplets of suspended cells
with droplets of cell suspension medium. In this way, concentration
of cells is reduced by the ratio of the combined volumes. Combined
droplet can be split in smaller droplets which can be further
merged with cell suspension medium for additional cell
concentration reduction. By repeating the procedure above, droplets
with single cells can be generated and used in single-cell
assays.
[0074] The results described above demonstrate that DMF can be used
to implement cell-based assays with very high performance. With
reduced reagent and cell consumption, and automated liquid
manipulation, DMF devices outperformed standard well plate assays,
and resulted in significant improvements in assay sensitivity. The
above results clearly demonstrate the efficacy of c DMF cell-based
assays for phenotypic screening.
Cells in Suspension Culture
[0075] Cell culture entails growing cells in a growth medium under
controlled temperature and atmosphere conditions. For example,
mammalian cells are grown in humidified atmosphere at 37.degree. C.
and 5% CO.sub.2, in cell culture incubators. Growth medium supplies
nutrients and growth factors to cells; its ingredients are cell
type dependant. In standard cell culture, cells grow suspended in
milliliter volumes in cell culture flasks; they are
split/subcultured every 2-3 days and resuspended in a fresh growth
medium.
[0076] In one embodiment of this invention we demonstrate: (1)
growing cells in nanoliter-microliter droplets in DMF devices (in a
cell culture incubator), (2) changing media daily, and 3) splitting
cells every 2-3 days. Media change involves adding one or more
droplets of fresh media to a droplet of incubated cells and thereby
partially replenishing growth media. Cells are further incubated in
the combined droplet or in smaller droplets generated by splitting
the combined droplet. Cell subculture or splitting is achieved
similarly to media change by combining (merging and mixing) a
droplet of incubated cells and a droplet of fresh media, splitting
the combined droplet, and repeating this procedure using the split
droplet(s) until a desired cell concentration is reached. Final
droplets are then incubated, while other droplets of suspended
cells generated in the subculturing process are discarded.
Multiplexed Cell Culture/Cells Assays
[0077] In a multiplexed assay 100 (shown in FIG. 10), multiple
droplets 106 containing one kind or multiple kinds of cells are
exposed to droplets 108 containing one or multiple reagents 104 and
are assayed similarly to the assays described above. Cells in a
suspension and cell-assay reagents can be deposited in the device
either by dispensing them from device reservoirs 102 (cells) and
104 (reagents) or by dispensing them using external means (e.g.,
pipette, robotic dispenser, etc.), not shown herein. A multiplex
device, an example of which is shown in FIG. 10, can also be used
for multiplex cell culture, where cells can be grown and maintained
in multiple droplets.
[0078] There are several ways of configuring the reservoirs. In one
configuration of the method and system the reservoirs may be
external to digital microfluidic device and include for example
arrays of pipettes, robotic dispensers, microprinters and
microstamps. Alternatively, the reservoirs could be integrated as
part of the digital microfluidic device, which are in flow
communication with the hydrophobic/dielectric surface above the
array of actuating electrodes. The reservoirs can be containers
integrated as part of the digital microfluidic device.
Alternatively they may include actuating electrodes from said array
of actuating electrodes modified to act as the liquid reservoirs as
shown in FIG. 1 where reservoir electrodes 32, 34, and 36 store
droplets 42, 44, 46 containing cells, reagent, and dye,
respectively.
[0079] The reservoirs could be part of a cartridge assembled with
the digital microfluidic device which is in flow communication with
the hydrophobic/dielectric surface above the array of actuating
electrodes.
[0080] The droplets are then translated to pre-selected sites on
the top surface of the substrate 114 on which the array of
actuating electrodes 116 is located. Assays in multiple droplets
are performed simultaneously or sequentially in a certain order
defined by the cell assay protocol. For example, a computer
controller interfaced to the device reservoirs and associated
dispensing devices is programmed to dispense droplets of the
suspension of cells and droplets of one or more cell assay reagents
onto the top surface of the dielectric layer covering the electrode
array 116 and surface of the substrate 114, and translating them
over said array of actuating electrodes for mixing the droplets in
selected positions on the array of actuating electrodes to form one
or more secondary droplets in a selected order defined by a
selected cell assay protocol for which said computer controller is
programmed.
[0081] Signals from secondary droplets are detected using
multiplexed detection instruments such as optical sensors, optical
detectors comprising a light source and a photodetector, optical
detectors that measure absorbance, fluorescence, epifluorescence,
chemiluminescence, UV light detector, radiometric detector,
scanning, imaging, and confocal microscopy detectors, CCD cameras,
and microplate readers. The detection step is to detect or identify
any reaction products formed by the cell assay, or to identify,
monitor and count the cells if a cell culture is being performed to
mention just a few.
[0082] The detection step may be conducted by first translating the
secondary droplet(s) to one or more selected positions on the
substrate surface for analysis or the secondary droplet(s) may be
removed from the device and analyzed externally.
[0083] All waste liquid droplets generated during the assay are
translated to the waste container 120. Reservoirs 122 may contain
wash solutions for cleaning the surface of the device between
assays.
Experimental
[0084] The use of the digital microfluidics for conducting
droplet-based cell assays using digital microfluidics will now be
illustrated with the following non-limiting examples/studies. More
particularly, herebelow, it is shown experimentally that the
effects of actuation by digital microfluidics on cell vitality are
minimal, and in addition, it is shown that a cytotoxicity assay
implemented by DMF has much better sensitivity than macroscale
methods, which suggests applications in regulatory policy and in
drug discovery. It is also demonstrate compatibility of DMF cell
assays with fluorescence microplate reader detection. This
technique has great potential as a simple yet versatile analytical
tool for implementing cell-based assays on the microscale.
Reagents and Materials.
[0085] Unless otherwise indicated, reagents used outside of the
clean room were purchased from Sigma-Aldrich (Oakville, ON), and
cells and cell culture reagents were from American Type Culture
Collection (ATCC, Manassas, Va.). Fluorescent dyes were from
Invitrogen-Molecular Probes (Eugene, Oreg.), Parylene-C dimer was
from Specialty Coating Systems (Indianapolis, Ind.), and Teflon-AF
was purchased from DuPont (Wilmington, Del.). Clean room reagents
and supplies included Shipley S1811 photoresist and MF-321
developer from Rohm and Haas (Marlborough, Mass.), solid chromium
and gold from Kurt J. Lesker Canada (Toronto, ON), standard gold
etchant from Sigma-Aldrich, CR-4 chromium etchant from Cyantek
(Fremont, Calif.), AZ-300T photoresist striper from AZ Electronic
Materials (Somerville, N.J.), and hexamethyldisilazane (HMDS) from
Shin-Etsu MicroSi (Phoenix, Ariz.). Concentrated sulfuric acid and
hydrogen peroxide (30%) were from Fisher Scientific Canada (Ottawa,
ON), and piranha solution was prepared as a 3:1 (v/v) mixture of
sulfuric acid and hydrogen peroxide.
Cell Culture.
[0086] Jurkat T-cells (human leukemia lymphocytes) were maintained
in a humidified atmosphere (5% CO.sub.2, 37.degree. C.) in RPMI
1640 medium supplemented with 10% fetal bovine serum (Invitrogen
Canada, Burlington, ON), penicillin (100 IU/mL), and streptomycin
(100 .mu.g/mL). Cells were subcultured every 3-4 days at
.about.1.times.10.sup.6 cells/mL. A working buffer of 0.2% (wt/v)
pluronic F68 (Sigma-Aldrich) in Dulbecco's phosphate buffered
saline (PBS) (Invitrogen Canada) was used for most cell-based
assays. Prior to experiments, cells were washed three times in PBS,
suspended in 0.2% F68 (wt/v) in PBS at 3.5.times.10.sup.6 cells/mL,
and then incubated at room temperature (1 h). Cell numbers and
viability were quantified using a hemocytometer and trypan blue
exclusion (Invitrogen Canada) immediately prior to all experiments.
Prior to cell viability/proliferation assays and analysis by mass
spectrometry, cells were incubated for 1 h in 3% (wt/v) F68 in PBS
at 7.2.times.10.sup.6 cells/mL and at 6.times.10.sup.7 cells/mL,
respectively.
Device Fabrication and Use.
[0087] Digital microfluidic devices were fabricated using
conventional microfabrication methods. 100 nm thick gold electrodes
were patterned on the bottom plate of a device (glass wafer) and
coated with 2 .mu.m of Parylene-C and 50 nm of Teflon-AF.
Unpatterned indium-tin oxide (ITO) coated glass substrates were
coated with 50 nm of Teflon-AF. Devices were assembled with an
unpatterned ITO-glass top plate and a patterned bottom plate and
separated by a .about.150 .mu.m thick spacer. Driving potentials
(100-140 V.sub.RMS) were generated by amplifying the output of a
function generator operating at 15 kHz. Droplets were sandwiched
between the two plates and actuated by applying driving potentials
between the top reference electrode 22 and sequential electrodes 14
on the bottom plate (FIG. 2(a)) via the exposed contact pads.
Droplet actuation was monitored and recorded by a CCD camera mated
to a stereomicroscope with fluorescence imaging capability. Most
devices used here had a geometry identical to that shown in FIG.
2(a) (or FIG. 1), with 1 mm.times.1 mm actuation electrodes
(suitable for manipulating 150 nL droplets), and inter-electrode
gaps of 5-40 .mu.m. The reservoirs were 2 mm.times.2 mm electrodes.
Some devices had 7 mm.times.7 mm actuation electrodes which were
used to manipulate much larger droplets (11 .mu.L).
Electrical Field Modeling.
[0088] Electrical fields in digital microfluidic devices were
modeled with COMSOL Multiphysics 3.3a (COMSOL, Burlington, Mass.)
using the conductive media direct current module and the
electrostatics module, shown in FIG. 4. The two-dimensional
geometry of the model was nearly identical to the device
illustrated in FIG. 2, including three patterned electrodes (1 mm
length) on the bottom plate, a layer of Parylene-C (2 .mu.m thick),
a layer of PBS and air (150 .mu.m thick), and a continuous
electrode on the top plate. The hydrophobic Teflon AF layer 18 was
omitted from the model because of its porosity and insignificant
thickness. Dielectric constants, .epsilon., and conductivities,
.sigma., used in the model included .epsilon..sub.parylene=2.65,
.epsilon..sub.pbs=70, .epsilon..sub.air=1, .sigma..sub.parylene=0
S/m, .sigma..sub.air=0 S/m, and .sigma..sub.pbs=4.7 S/m (measured
using a conductivity meter). With a 100 V potential applied between
the bottom-right electrode and the top electrode (ground), a mesh
with 233,831 triangular elements was used to simulate electrical
field, using the linear solver UMFPACK.
Vitality Assays.
[0089] The effects of the electric field driven droplet actuation
on cell vitality were evaluated by three assays, measuring cell
viability (FIG. 5 day 0), proliferation (FIG. 5 day 2), and
biochemistry (FIG. 6). In these vitality assays, large droplets
(>1 .mu.L) were used because the more conventional
sub-microliter droplets (used in the cell phenotype assays) were
difficult to handle off-chip and did not contain enough cells for
analysis. In the cell viability and proliferation assays, ten 11
.mu.L droplets of cells suspended in PBS/F68 (each containing
.about.79,200 cells) were actuated on devices with 7.times.7 mm
electrodes. Each droplet was moved across 10 electrodes
(approximately 15 s of actuation per droplet) and was then removed
from the device and suspended in 300 .mu.L of cell medium at
2.5.times.10.sup.5 cells/mL. For viability assays, immediately
after suspension in media, live and dead cells were counted on a
hemacytometer with trypan blue exclusion. For proliferation assays,
live and dead cells were counted after 48 h of incubation off-chip
(humidified incubator, 5% CO.sub.2, 37.degree. C.). A second group
of ten 11 .mu.L droplets of the original cell solution (in PBS/F68)
were treated identically, but were not actuated, and served as a
control. The data was analyzed with two-tailed t-test assuming
unequal variances.
[0090] In the cell biochemistry assay, four 11 .mu.L droplets of
cell suspension (.about.6.6.times.10.sup.5 cells/droplet) were
actuated over ten electrodes as above, and were then pooled and
suspended in lysing medium at 3.times.10.sup.7 cells/mL. Lysing
medium was PBS with 3% (wt/v) F68, 1% Triton X-100, and 1 mM
phenylmethylsulphonyl fluoride (PMSF). After incubation on ice (30
min), the lysate was centrifuged (12,000 rpm, 5 min) and the
supernatant was collected and stored in a -85.degree. C. freezer.
Immediately prior to analysis, the supernatant (100 .mu.L) was
thawed and desalted using a microspin G-25 column (Amersham
BioSciences, Piscataway, N.J.) at 2800 rpm for 2 min. Proteins were
eluted in distilled water with 0.05% (v/v) Kathon (1.5 .mu.L), and
the eluent was spotted onto a MALDI (matrix assisted laser
desorption/ionization) target plate. A 1.5 .mu.L aliquot of MALDI
matrix solution (10 mg/mL sinapinic acid in 80% (v/v)
acetonitrile/water) was added and the combined droplet was allowed
to dry. Non-actuated droplets of the original cell suspension were
lysed and processed identically, and served as a control.
[0091] Samples were analyzed using a MALDI-TOF Micro MX mass
spectrometer (Waters, Milford, Mass.) in linear positive mode for
the mass range of 4,000 to 25,000 m/z. One hundred shots were
collected per spectrum, with laser power tuned to optimize the
signal over noise ratio. Data were then processed by normalization
to the largest analyte peak, baseline subtraction, smoothed with a
15-point running average.
Cell Phenotype Assays.
[0092] For phenotypic assays, cells were exposed to the surfactant,
Tween 20 (lethal to mammalian cells at high concentrations),
diluted in working buffer in a range of concentrations (0.002% to
0.5% (wt/vol)). Each Tween 20 concentration was evaluated in 4-6
replicates. In each experiment, a 150 nL droplet containing
.about.525 cells was dispensed and merged with a 150 nL droplet
containing Tween 20. The merged droplets were then actively mixed
by moving them on four neighboring electrodes in a circle. After 20
min of incubation in a humidified environment (a closed petri dish
half-filled with water), the combined droplet containing cells and
Tween 20 was merged and mixed with a 150-nL probe droplet
containing viability dye(s), and then incubated for a second time
in a humidified environment (20 min). In all experiments, the probe
droplet contained calcein AM (1 .mu.M in the working buffer), and
in some experiments, the droplet also contained ethidium
homodimer-1 (2 .mu.M in the working buffer).
[0093] For quantitative experiments, a digital microfluidic device
was positioned on the top of a well plate and inserted into a
fluorescence microplate reader (Pherastar, BMG Labtech, Durham,
N.C.) equipped with a module for 480 nm excitation and 520 nm
emission. Each droplet was evaluated using a multipoint scanning
program, in which the average fluorescence was recorded from each
of 9 excitation flashes illuminated onto a 1-mm square 3.times.3
array with 0.5 mm resolution. The array was located in the centre
of each droplet, and the focal height was set for each analysis at
the highest-signal intensity, with gain=376. This multipoint
program, designed by BMG Labtech for standard assays in well
plates, was found empirically to have lower variance between runs
than comparable single point analyses. Samples containing only
Tween 20, pluronic F68, and calcein AM in PBS were evaluated to
determine the background signal. Each analysis was repeated 4-6
times to determine standard deviations. All data were normalized to
the average fluorescence intensity of cell samples exposed to
control droplets (containing no Tween-20), and were plotted as a
function of Tween-20 concentration.
[0094] For comparison, each assay implemented by digital
microfluidics was duplicated in standard 384-well plates by
pipetting reagents, cells, and dyes. In these experiments, all
parameters were identical to those described above, except that the
.about.525 cells, reagents, and dyes were suspended in a final
volume of 15 .mu.L.
Culturing and Assaying Adherent Cells
[0095] The majority of mammalian cells are adherent, i.e. anchorage
dependent. In a further embodiment of the present invention, we
demonstrate that DMF can also be used to culture and assay adherent
cells. In in vitro conditions, adherent cells grow in layers
attached to a substrate that is typically hydrophilic and
negatively charged, such as tissue culture treated polystyrene.
Cells are maintained/grown in cell culture (growth) media in
incubators with humidified atmosphere at 37.degree. C. and with 5%
CO.sub.2.
[0096] As shown in FIGS. 11a, 11b, 11c, and 11d, the surface of a
DMF device 200 (specifically the hydrophobic surface 18 that covers
the dielectric material 16 on the lower electrode 14 (see FIG.
2(a)) is modified in specific areas, cell culture sites (CCS) 202,
to facilitate cell adhesion and proliferation (cell growth and
division). The surface modification procedure reported here makes
use of standard techniques, such as depositing (microprinting,
micorstamping) a bio-substrate (typically extracellular matrix
proteins 206), rendering a hydrophilic and charged surface via
microfabrication, or any other surface modification procedure that
can also be cell specific.
[0097] In addition to using standard techniques, a bio-substrate
can be formed by dispensing a droplet of a bio-substrate solution
in a DMF device and translating it to the cell culture site 202,
where after incubation and drying, it forms a bio-substrate layer
for cell attachment. In this case, a device has an extra reservoir
holding the bio-substrate solution. After the cell culture site 202
is formed, cells are seeded by generating a droplet 214 of growth
media with suspended cells 212 on the cell culture site CCS 202
(FIG. 11c). Cells are allowed to adhere to the surface forming a
cell monolayer 204 (FIG. 11d).
[0098] There are two ways of generating a droplet 214 on the cell
culture sites 202: (1) by actively dispensing a droplet from a
device reservoir or via external means (e.g. pipetting) and
translating the droplet to the cell culture sites 202 (FIG. 11(a)),
and (2) by actuating a droplet 216 (source droplet) larger than the
cell culture sites 202 over the cell culture sites 202 and thereby
passively dispensing the desired droplet on the hydrophilic cell
culture sites 202 (FIG. 11(b)). Passive dispensing will be
described in more details in the following section.
Passive Dipensing, Passive Washing, Passive Media/Reagent
Exchange
[0099] Referring to FIG. 12, when a source droplet 210 is actuated
in a DMF device over a patterned hydrophilic area 201 smaller than
the base area of the source droplet 210, it leaves behind a smaller
droplet 230 on the hydrophilic area 201 and the rest of source
droplet 210 is translated away from droplet 230. This method of
generating droplets is termed passive dispensing. Methods for
producing the hydrophilic areas 201 include but are not limited to
microfabrication techniques (e.g. exposing hydrophilic layers of a
device, such as glass or electrodes, in specific areas),
hydrophobic surface plasma treatment, or deposition of a thin,
patterned, hydrophilic layer onto a device surface. Hydrophilic
areas can be formed on either the top plate, the bottom plate, or
both the top and bottom plate of a two plate device. In the
applications disclosed herein of adherent cell culture and
assaying, hydrophilic areas 201 are used as the cell culturing
sites (indicated by reference numeral 202 in FIG. 11) which
preferably patterned by depositing bio-substrates, made from cell
specific constituents, such as, but not limited to, extracellular
matrix (ECM) proteins. ECMs are more favorable substrate for cell
attachment than bare glass, electrodes, or a dielectric layer.
[0100] Examples of extracellular matrix proteins include, but are
not limited to fibronectin, laminin, collagen, elastin. The cell
specific constituents may also comprise synthetic molecules
comprised of one of poly-L-lysine, poly-D-lysine and any
combination thereof for example.
[0101] Typically, there are no electrodes underneath hydrophilic
areas, as these areas (inherently hydrophilic) do not need to be
electrically addressed to attract droplets; however, they have to
be at least in the vicinity of electrodes. It will be appreciated
that the hydrophilic arrays can also be formed on the top surface
of the layer coating electrodes right above electrodes themselves.
In most cell-based applications, it is desirable to have
transparent attachment substrate to enable facile cell
visualization.
[0102] Referring to FIG. 13, the size and position of a hydrophilic
area can vary relative to size and position of electrodes 14 for
source droplets actuation. Two relative sizes of hydrophilic
areas--1/4 and 1/9 of the electrode size were studied, and several
positions relative to electrodes 14 and to a source droplet path.
It should be noted that size and position of hydrophilic areas 201
is not limited by the examples in FIG. 13, and that the shape of
hydrophilic areas 201 and actuating electrodes 14 is not limited to
the square shape.
[0103] Referring to FIG. 14, when a hydrophilic area 201 is already
occupied by a droplet 230, a source droplet 210 will remove the
smaller droplet 230 and replace it with a new droplet 232 of the
source solution while removing droplet 230 in droplet 210'. This
process is termed passive washing or passive exchange of liquid
solutions on hydrophilic areas 201 (e.g., on CCSs) in a DMF device.
We report passive exchange efficiency of .gtoreq.95% with a single
source droplet, or .gtoreq.99% with two or more consecutive source
droplets. FIG. 15 shows efficency of 0.5 nM fluorescein passive
exchange with phosphate buffered saline. These results were
obtained with fibronectin hydrophilic areas 201, .about. 1/9 of the
electrode size, having two different positions relative to
actuating electrodes 14.
Culturing and Passaging Adherent Cells
[0104] For adherent cell culture, a DMF device with seeded cells is
placed in a cell culture incubator and a droplet of culture media
on top of the cell layer 204 is regularly replenished with fresh
media via DMF passive exchange every 24 h. We report culturing
cells on cell culture sites 202 for 72 h; growth characteristics
and morphology of the cells are comparable to cells grown in
standard tissue culture flasks (FIG. 16). No detachment of cells
was observed during media droplet actuation over the cell culture
sites 202. Cells are subcultured at regular intervals using
standard subculturing protocols adapted to DMF system: (1) washing
cells as shown in FIG. 17(b) in which washing droplet 213 has been
dispensed and translated over cell culture site 202, (2) harvesting
cells by dispensing and translating a droplet 215 containing a
dissociation agent (e.g. trypsin, collagenase) over cell culture
site 202 as shown in FIG. 17(c) and incubating to detach the
adhered cells and resuspend them as shown in FIG. 17(d), (3) a
droplet 240 containing a blocking agent (typically serum in cell
culture media) for blocking the dissociation agent is dispensed and
translated over cell culture site 202, while removing the detached
cells away from the cell culture sites 202 as shown in FIG. 17(e),
(4) splitting the resulting cell suspension as necessary and
resuspending in fresh media in droplet 242 and (5) seeding
resuspended cells on a new cell culture site 202 as shown in FIG.
17(f). Blocked dissociation agent and cell suspension are diluted
in a big source droplet 240 of a blocking agent (cell culture media
with serum) by the ratio of the volumes of the two droplets, cell
culture site 202 droplet and the source droplet. In step (4), the
resulting cell suspension can be split in smaller droplets and
resuspended in droplets of fresh media for further reduction of
cell concentration. When a desired cell concentration is achieved,
new generation of cells is seeded on new cell culture sites 202 by
either translating actively dispensed droplets of the cell
suspension to new cell culture sites, or by passively dispensing
droplets with cells on cell culture sites 202 from droplet 242
(FIG. 17f). The inventors have demonstrated subculturing several
generations of mammalian cells in the same DMF device following the
procedure outlined above.
Assaying Adherent Cells
[0105] Adherent cell assays in DMF devices are executed in droplets
on cell culture sites 202 where adherent cells are seeded. Devices
with seeded cells are placed in incubators for few hours or
overnight to allow cell attachment and adjustment to a new DMF
device environment (FIG. 18a). When adherent cell deposits 204 are
ready for assaying, droplets of reagents and washing solutions are
deposited on cell culture sites 202 either by translating a droplet
actively dispensed from a device reservoir or externally, or by
passive dispensing/exchange from source droplets 250 (FIG. 18b).
Source droplets 250 are either dispensed via DMF from reservoirs or
externally deposited on a device. Washing solutions and reagents
are incubated with cells following cell assay protocols (FIG. 18c).
Upon assay completion, cell response to a stimulus (e.g. a lead
drug compound) can be detected and measured by apparatus 260 which
may be any standard means (e.g. fluorescence microscopy, microplate
reader to give a few examples) (FIG. 18d).
[0106] In assays targeting extracellular biochemistry (growth
factors, signaling molecules, metabolic products, etc.), cell
response to stimulus is detected in medium where cells are grown
and stimulated with reagents, rather than in cells. Medium can be
analyzed by immunoassays or other means. Droplets of cell
suspension can alternatively be removed from the cell culture sites
202 (e.g. with a bigger source droplet) and its signal can be
detected on another spot or its contents can be analyzed
externally.
Multiplexed Adherent Cell Culture/Cell Assays
[0107] Referring to FIG. 19, multiple cell culturing sites 202 in a
DMF device 300 which is similar to device 100 in FIG. 10 but device
300 includes a plurality of cell culture sites 202. Device 300 may
be used in multiplexed assays where cells of one kind or multiple
kinds are assayed with one or multiple reagents simultaneously in
which cell culturing may be involved as well. In addition, a single
cell culture site 202 can be seeded with multiple cell lines (cell
co-culture). Assay reagents and/or culture media can be delivered
to cell culture sites 202 via passive dispensing/exchange or in
actively dispensed droplets.
[0108] In a multiplexed assay, a single source droplet can deliver
reagents to multiple cell culture sites 202 (serial passive
dispensing/exchange), or to only one cell culture site 202
(parallel passive dispensing/exchange). Signals from assayed cells
or suspension media is detected using multiplexed detection
instruments such as microplate readers.
Experimental
[0109] The following non-limiting examples demonstrates the
efficacy of the present invention for conducting adherent cell
assays and culture.
Device Design and Fabrication.
[0110] Digital microfluidic devices were fabricated using
conventional microfabrication methods. 100 nm thick gold electrodes
were patterned on the bottom plate of a device (glass wafer) and
coated with 2 .mu.m of Parylene-C and 50 nm of Teflon-AF.
Unpatterned indium-tin oxide (ITO) coated glass substrates were
coated with 50 nm of Teflon-AF. Devices were assembled with an
unpatterned ITO-glass top plate and a patterned bottom plate and
separated by a .about.150 .mu.m thick spacer. Driving potentials
(100-140 V.sub.RMS) were generated by amplifying the output of a
function generator operating at 15 kHz. Droplets were sandwiched
between the two plates and actuated by applying driving potentials
between the top reference electrode 22 and sequential electrodes 14
on the bottom plate (FIG. 2(a)) via the exposed contact pads. Most
devices had a basic geometry identical to that shown in FIG. 11
with the addition of reservoirs. Source droplets (.about.800 nL)
were actuated on 2.5 mm.times.2.5 mm actuation electrodes, and
smaller droplets were actuated on 0.8 mm.times.0.8 mm actuation
electrodes. Cell culture site (CCS) areas were patterned either as
transparent, non-conductive fields in 2.5 mm.times.2.5 mm
electrodes or as smaller (0.8 mm.times.0.8 mm) electrodes within
the area of larger 2.5 mm.times.2.5 mm electrodes. Devices were
sterilized in 70% ethanol prior to use.
Cell Culture
[0111] NIH-3T3 cells (mouse fibroblasts) were maintained in a
humidified atmosphere (5% CO.sub.2, 37.degree. C.) in DMEM
supplemented with 10% fetal bovine serum, penicillin (100 IU
mL.sup.-1), and streptomycin (100 .mu.g mL.sup.-1). Cells were
subcultured every 2-3 days at 5.times.10.sup.3 cells cm..sup.-2
Prior to each DMF experiment, cells were suspended in DMEM with the
addition of 0.05% (wt/v) pluronic F68 (Sigma-Aldrich) at
.about.7.times.10.sup.5 cells mL..sup.-1 Cell number and viability
were quantified using a hemocytometer and trypan blue exclusion
(Invitrogen Canada) immediately prior to all experiments.
DMF Cell Seeding
[0112] CCSs were formed by depositing 500 nL droplets of
fibronectin (100 .mu.g mL.sup.-1 in ddH.sub.2O) on designated areas
in DMF devices. Fibronectin solution was air-dried resulting in
.about.1 mm.sup.2 bio-substrates with .about.5 .mu.g/cm.sup.2 of
fibronectin. Cell suspension was delivered to CCSs by either
passive dispensing from a source droplet or by translating actively
dispensed droplets from a device reservoir to CCSs. CCS droplets
were .about.200 nL in volume and contained .about.140 cells. Cells
were allowed to attach to the substrate and adapt overnight in a
cell culture incubator (5% CO.sub.2, 37.degree. C.).
DMF Cell Culture
[0113] NIH-3T3 cells were maintained on CCSs by changing media via
passive dispensing every 24 hours. Complete DMEM containing 0.05%
(wt/v) pluronic F68 was dispensed in .about.800 nL droplets and
translated over CCSs while replenishing CCS droplet of media.
Complete media exchange was accomplished with two consecutive
source droplets and cells were returned to the incubator. No cell
detachment was observed during passive media exchange.
DMF Cell Subculture
[0114] Upon reaching confluency on CCSs, cells were subcultured
following standard subculturing protocols adapted to the DMF
format. All reagents and media containing 0.05% (wt/v) pluronic F68
were delivered to cells using passive dispensing/exchange from two
consecutive source droplets. Cells were first washed with PBS
without Ca.sup.2+/Mg.sup.2+ and then supplied and incubated with
GIBCO Trypsin-EDTA dissociation agent (0.25% Trypsin, 1 mM EDTA
4Na) for 5-10 min at 37.degree. C. DMEM source droplet was then
translated to the CCS to block the dissociation agent with the
serum present in media, whereby harvested cells were resuspended in
DMEM droplet at the 1:4 ratio. DMEM droplet with suspended cells
was actuated away from the CCS and used either as a source droplet
or a reservoir droplet to seed the new generation of cells on a new
CCS in the same device. Seeded cells were placed in a cell culture
incubator overnight followed by media change. Cells were grown on
the new CCS for 2 days and further subcultured on the same
device.
DMF Cell Viability Assay
[0115] Cells cultured on CCSs were assayed on a device for
viability. Source droplets of 0.05% (wt/v) pluronic F68
(Sigma-Aldrich) in phosphate buffered saline containing viability
dyes, calcein AM (1 .mu.) and ethidium homodimer-1 (2 .mu.M)
(Invitrogen Canada), were dispensed in a device and translated over
the CCS. With two consecutive source droplets, growth media was
removed from the CCS and replaced with viability dyes. Cells were
incubated with dyes at room temperature and visualized using
stereomicroscope. Viability of cells was higher than 95% and there
was no significant difference in morphology between cells grown on
CCSs and cells grown in cell culture flasks.
[0116] It will be understood that when doing cell culturing or cell
assaying, the suspension of cells may contain a combination of
cells, a suspension medium, and a non-ionic surfactant. The
suspension medium may be selected to facilitate cell-containing
droplet actuation by preventing non-specific adsorption of cells
and proteins to device surfaces. The suspension of cells may be a
combination of cells and a suspension medium comprised of block
copolymers formed from poly(propylene oxide) and poly(ethylene
oxide), pluronic F68, pluronic F127, hydrophilic polymers, sodium
bicarbonate, phosphate buffered saline (PBS), HEPES, and other
biological buffers, and any combination thereof, which may be
combined or mixed with cell culture medium which in turn may
include balanced salt solutions, nutrient mixtures, basal media,
complex media, serum free media, insect cell media, virus
production media, serum, fetal bovine serum, serum replacements,
antibiotics, antimycotics, and any combination thereof.
[0117] In an embodiment the suspension of cells may be a
combination of cells, phosphate buffered saline, and pluronic F68.
The droplets including a cell assay reagent may include chemicals,
biochemicals, drugs, drug lead compounds, toxins, surfactants,
transfection reagents, supplements, cell culture media,
anti-clumping agents, streptavidin, biotin, antibody production
enhancers, antibodies, antibody ligands, nucleic acids, nucleic
acid binding molecules, enzymes, proteins, viruses, cell process
agonists or antagonists, labeling agents, fluorescent dyes,
fluorogenic dyes, viability dyes, calcein AM, quantum dots, nano
particles, Tween 20, and ethidium homodimer-1, block copolymers
formed from poly(propylene oxide) and poly(ethylene oxide),
pluronic F68, pluronic F127, hydrophilic polymers, sodium
bicarbonate, phosphate buffered saline (PBS), HEPES, and other
biological buffers, and any combination thereof, which may be
combined or mixed with cell culture medium which in turn may
include balanced salt solutions, nutrient mixtures, basal media,
complex media, serum free media, insect cell media, virus
production media, serum, fetal bovine serum, serum replacements,
antibiotics, antimycotics, and any combination thereof.
[0118] The cells in the suspension of cells may include
primary/isolated or transformed/cultured cells selected from the
group consisting of various eukaryotic and prokaryotic cells,
including animal cells (blood cells, human leukemia cells,
lymphocytes, beta cells, oocytes, eggs, primary cells, primary bone
marrow cells, stem cells, neuronal cells, endothelial cells,
epithelial cells, fibroblasts), insect cells, plant cells,
bacterial cells, archebacterial cells.
[0119] As used herein the word "incubation" can mean allowing a
reaction to take place over a period of time under specified
conditions. For cell assays involving mixing of cells with one or
more cell assay reagents, the incubation period may be very short
or almost instantaneous upon mixing the droplets wherein the
reaction or response of the cells to the reagent occurs quickly.
For cell culture, "incubation" can mean maintaining the cells
growing or alive under specific conditions and the period of time
of the "incubation" may be arbitrary, after which point the cells
may be subcultured, assayed or subject to further culturing.
[0120] The results disclose herein demonstrate the utility of the
present invention for its application of digital microfluidics to
multiplexed, high throughput, phenotypic cell-based assays, an
important tool used in drug discovery and environmental monitoring.
To facilitate high-throughput screening, arrays of DMF cell culture
sites (FIG. 19) can be addressed with compounds from chemical
libraries, and the potential drugs evaluated on the basis of
observed phenotypic changes. The proposed method will enable
high-throughput phenotypic screening with 100-1000.times. lower
reagent consumption than conventional methods; in addition, the
devices are inexpensive (relative to robotic dispensers), have
small laboratory footprint and no moving parts. This method could
transform high-throughput screening, making it attractive to
pharmaceutical companies and accessible for basic and applied
scientists, world-wide.
[0121] In addition to cell assaying the inventors disclose herein
the first multigenerational lab-on-a-chip cell culture using DMF
devices. Cells are grown, maintained and subcultured in nanoliter
volumes. DMF devices are inherently easily automated and as such
have a high potential to be used as tool for a completely automated
microscale cell culture system.
[0122] As used herein, the terms "comprises", "comprising",
"includes" and "including" are to be construed as being inclusive
and open ended, and not exclusive. Specifically, when used in this
specification including claims, the terms "comprises",
"comprising", "includes" and "including" and variations thereof
mean the specified features, steps or components are included.
These terms are not to be interpreted to exclude the presence of
other features, steps or components.
[0123] The foregoing description of the preferred embodiments of
the invention has been presented to illustrate the principles of
the invention and not to limit the invention to the particular
embodiment illustrated. It is intended that the scope of the
invention be defined by all of the embodiments encompassed within
the following claims and their equivalents.
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